Kojima S, Kuriki Y, Sato Y, Arisaka F, Kumagai I, Takahashi S, Miura K
Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan.
Biochim Biophys Acta. 1996 May 23;1294(2):129-37. doi: 10.1016/0167-4838(96)00003-9.
Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.
设计并表征了两种被认为能形成α-螺旋的肽。一种是“α(3)-肽”，由21个残基组成，包含七残基序列Leu-Glu-Thr-Leu-Ala-Lys-Ala的三个重复。由于Leu残基的疏水表面以及Lys和Glu残基的亲水表面，该肽似乎具有两亲性，从而形成束状结构。另一种是“α(3)-GPRRG-α(3)肽”，有47个残基，其中两个α(3)-肽通过五残基序列Gly-Pro-Arg-Arg-Gly连接。编码这些肽的基因通过甲硫氨酸密码子与腺苷酸激酶基因融合。所得融合蛋白以包涵体形式表达，用溴化氰切割后纯化肽。然后通过测量其圆二色光谱来检测肽在各种缓冲液中的稳定性。α(3)-肽显示出α-螺旋的浓度依赖性稳定。沉降平衡超速离心表明它形成了由四条多肽链组成的束状结构，分析凝胶过滤还检测到寡聚化过程中的二聚体中间体。将pH值调至2或12时，α(3)-肽的稳定性降低，这是由于带电残基的静电排斥。因此，通过高盐浓度掩盖排斥作用，α(3)-肽在增加离子强度时，尤其是在酸性或碱性缓冲液中得到稳定。在中性pH和高盐浓度的缓冲液中，高浓度的α(3)-肽形成可见聚集体，这可能是由于α-螺旋束暴露的疏水表面。另一方面，α(3)-GPRRG-α(3)肽未显示出浓度依赖性的可逆解离和缔合。即使在低浓度下，它也以三聚体形式存在，表明α(3)-GPRRG-α(3)肽的缔合非常紧密。与α(3)-肽相反，α(3)-GPRRG-α(3)肽在各种pH值和盐浓度下都非常稳定。这似乎是由于七残基重复次数从三个增加到六个导致疏水相互作用增强，尽管每组三个重复之间由一个五残基序列隔开。
